Experimental Methods for Protein Structure Determination
59 Questions
0 Views

Choose a study mode

Play Quiz
Study Flashcards
Spaced Repetition
Chat to lesson

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

Which of the following is NOT a recommended statistic to report for assessing structural quality?

  • Number of amino acids in the sequence (correct)
  • Precision of structures: RMSD
  • Maximum restraint violation
  • Number of torsion angle restraints

    • The recommended goal for rmsd for backbone atoms is between 0.3-0.6 Å.

    True

    What is a very rough rule of thumb for assessing the equivalency of an NMR structure with respect to crystal structure?

    An NMR structure calculated with ≥20 restraints per residue is equivalent to a 2-2.5 Å crystal structure.

    The maximum restraint violation indicates the ___ of the structural restraints used.

    <p>quality</p> Signup and view all the answers

    Match the following metrics to their corresponding quality ranges:

    <p>Restraints per residue = &gt; 18 Backbone rmsd = &lt; 0.3 Heavy-atom rmsd = &lt; 0.75 Ramachandran Plot quality (%) = &gt; 95</p> Signup and view all the answers

    What is considered a very high resolution for heavy-atom rmsd?

    <p>&lt; 0.75 Å</p> Signup and view all the answers

    Long range restraints are considered less important than medium range restraints for high-quality NMR structures.

    <p>False</p> Signup and view all the answers

    According to the 1998 IUPAC Task Force recommendations, what is the maximum allowable deviation from idealized geometry?

    <p>Unusual bond lengths or bond angles</p> Signup and view all the answers

    Which structural representation highlights the secondary structure elements of a protein?

    <p>Ribbon model</p> Signup and view all the answers

    The R-factor of 0.0 indicates total disagreement between the crystallographic model and the experimental data.

    <p>False</p> Signup and view all the answers

    What is the typical R-factor range for a well-determined protein structure?

    <p>0.15 to 0.20</p> Signup and view all the answers

    Cryo-EM is beneficial for examining __________ structures and conformational heterogeneous states.

    <p>non-crystalline</p> Signup and view all the answers

    Match the following quality parameters to their descriptions:

    <p>R-factor = Measure of agreement between model and data Rfree = Global measure calculated from random reflections RMSD = Indicates deviation from stereochemical standards Ramachandran plot = Percentage of residues with unusual φ/ψ angles</p> Signup and view all the answers

    Which representation shows all individual atoms of each amino acid?

    <p>Ball-and-stick model</p> Signup and view all the answers

    An Rfree value exceeding R by more than 7% may indicate model defects.

    <p>True</p> Signup and view all the answers

    What does a high RMSD value (>0.03 Å) indicate?

    <p>Errors in the model</p> Signup and view all the answers

    In a space-filling representation, each non-hydrogen atom is represented as a sphere of its __________ radius.

    <p>van der Waals</p> Signup and view all the answers

    What is the purpose of polyethylene glycol in protein purification?

    <p>To reduce protein solubility and create a supersaturated state</p> Signup and view all the answers

    Which method directly images individual molecules using electron microscopy?

    <p>Cryo-EM</p> Signup and view all the answers

    The asymmetric unit is the largest portion of a crystal structure.

    <p>False</p> Signup and view all the answers

    What method is used to create protein crystals by vapor diffusion?

    <p>Hanging-drop method</p> Signup and view all the answers

    The formula for Bragg’s law is ___ + ___ = 2d·sinθ = n·λ.

    <p>BC, CD</p> Signup and view all the answers

    What indicates the presence of a constructive interference during X-ray diffraction?

    <p>Resulting spots on the detector</p> Signup and view all the answers

    Match the process with its description:

    <p>Vapor diffusion = Method for forming crystals by balancing concentrations X-ray diffraction = Technique used to analyze crystal structure Elastic scattering = Process where scattered waves maintain energy and wavelength Unit cell = Smallest repeating unit of a crystal structure</p> Signup and view all the answers

    A crystal is made from a few identical unit cells tightly packed together.

    <p>False</p> Signup and view all the answers

    What happens to the concentration of precipitant during the hanging-drop method?

    <p>It increases as water is lost from the drop.</p> Signup and view all the answers

    What primarily causes differences in resonance frequencies of protons?

    <p>Different chemical environments</p> Signup and view all the answers

    The 2D-NMR spectrum has resonances located only on the diagonal.

    <p>False</p> Signup and view all the answers

    What effect does the nuclear Overhauser effect (NOE) describe?

    <p>Dipole-dipole relaxation between protons in close proximity</p> Signup and view all the answers

    What does the presence of ill-defined regions in a protein structure usually indicate?

    <p>Conformational dynamics or lack of data</p> Signup and view all the answers

    The _______ technique detects neighbors of protons that are maximally separated by three bonds.

    <p>COSY</p> Signup and view all the answers

    Proteins can be considered static under normal conditions.

    <p>False</p> Signup and view all the answers

    Match the NMR techniques to their primary function:

    <p>COSY = Detects neighbors up to three bonds apart TOCSY = Detects entire amino acid spin systems NOESY = Measures spatial proximity of protons Scalar coupling = Coupling through covalent bonds</p> Signup and view all the answers

    Which approach is used for measuring distances in structure calculation?

    <p>Nuclear Overhauser Effect</p> Signup and view all the answers

    What can be confirmed by relaxation measurements?

    <p>Dynamics of proteins</p> Signup and view all the answers

    The size limitation in NMR-spectroscopy results in a maximum coverage of proteins with less than ___ amino acids.

    <p>250</p> Signup and view all the answers

    Spin-spin coupling primarily occurs between protons that are from different amino acids.

    <p>False</p> Signup and view all the answers

    What is a key characteristic of proton signals in a NOESY experiment?

    <p>They indicate protons in close spatial proximity.</p> Signup and view all the answers

    What is a consequence of larger protein sizes in NMR-spectroscopy?

    <p>Larger overlap of signals</p> Signup and view all the answers

    Match the molecular aspect with its corresponding feature:

    <p>Conformational dynamics = Indicates protein flexibility NMR-spectroscopy = Used for smaller proteins Disulfide bonds = Contributes to protein stability Relaxation measurements = Confirm dynamics of proteins</p> Signup and view all the answers

    The larger the coupling constants in scalar coupling, the __________ the magnetization transfer.

    <p>more efficient</p> Signup and view all the answers

    Excluding the highly disordered C-terminal region improves the fit of protein overlay analysis.

    <p>True</p> Signup and view all the answers

    What is the purpose of listing structural restraints in protein structure calculation?

    <p>To ensure structures are consistent with known data</p> Signup and view all the answers

    What is the primary challenge in analyzing larger proteins with NMR-spectroscopy?

    <p>Unambiguous assignment of resonances</p> Signup and view all the answers

    What does Bragg's law relate to in the context of diffraction?

    <p>The angle of diffraction</p> Signup and view all the answers

    The phase of a diffracted beam refers to its intensity only.

    <p>False</p> Signup and view all the answers

    What is the role of heavy atoms in the Multiple Isomorphous Replacement (MIR) method?

    <p>They enhance scattering and allow for phase calculation by binding to protein side chains.</p> Signup and view all the answers

    The electron density map is obtained through __________ transformation.

    <p>Fourier</p> Signup and view all the answers

    Match the following resolutions with their corresponding features:

    <p>~ 6 Å = Shape of the macromolecule &lt; 3 Å = Polypeptide chain tracing &lt; 1.5 Å = Individual non-hydrogen atoms resolved &lt; 1 Å = Location of hydrogen atoms</p> Signup and view all the answers

    Which technique uses synchrotron radiation to solve the phase problem?

    <p>Multiwavelength Anomalous Dispersion (MAD)</p> Signup and view all the answers

    The resolution value determines how separate the maxima appear in an electron density plot.

    <p>True</p> Signup and view all the answers

    What happens to the scattering when varying the wavelength around the absorption edge?

    <p>The contribution from the atoms to total scattering varies in amplitude and phase.</p> Signup and view all the answers

    The smallest distance of atoms that can appear as separate maxima is called __________.

    <p>resolution</p> Signup and view all the answers

    At what resolution can the positions of individual non-hydrogen atoms generally start to be resolved?

    <p>&lt; 1.5 Å</p> Signup and view all the answers

    Heavy metals are used in MIR because they have similar phase characteristics to proteins.

    <p>False</p> Signup and view all the answers

    What is the significance of the 0.77 Å resolution limit?

    <p>It is the physical limit for resolution when using copper Kα X-ray radiation.</p> Signup and view all the answers

    The phase problem can be solved using strategies like MIR and __________.

    <p>MAD</p> Signup and view all the answers

    Which of the following is NOT a property that defines each diffracted beam?

    <p>Color</p> Signup and view all the answers

    High-resolution structures give more confidence in the atom locations within the crystal.

    <p>True</p> Signup and view all the answers

    Study Notes

    Experimental Methods for Structure Determination

    • Methods for determining protein structure experimentally include X-ray crystallography, cryo-electron microscopy (EM), and nuclear magnetic resonance (NMR).
    • The Protein Data Bank (PDB) is a databank for experimentally determined 3D protein structures, supplemented by computed models.

    Protein Structures Determined by Experiments

    • Different experimental methods determine protein structures in different ways.
    • Experiments vary in how protein structures are determined by experiment.

    The Protein Data Bank (PDB)

    • The PDB is a comprehensive database of experimentally determined 3D protein structures.
    • It contains information about protein structures from X-ray crystallography, cryo-electron microscopy (EM), and nuclear magnetic resonance (NMR).
    • The PDB has recently expanded to include computed structure models from AI (e.g. AlphaFold).

    X-Ray Crystallography

    • X-ray crystallography is a method for determining the structure of proteins, and other molecules.
    • Protein crystallization is crucial for X-ray crystallography.
    • The process involves crystallizing the protein then irradiating the crystal with X-rays, recording the diffraction pattern, and calculating electron density maps to build an atomic protein model by iterative refinement. Crystallization parameters to produce protein crystals include type of solvent and precipitant, as well as the presence of ions and ligands (along with the pH and temperature of the solution).
    • Bragg's law and the phase problem are important concepts in X-ray crystallography
    • The obtained electron density map is used to build the 3D model of the protein.
    • The accuracy of the model is assessed using metrics like R-factor, Rfree factors, and the Ramachandran plot.
    • X-ray crystallography typically provides high-resolution structures for a well-determined protein structure.

    Cryo-Electron Microscopy (Cryo-EM)

    • Cryo-EM is a method for determining the structure of proteins in a solution using electron microscopy.
    • Cryo-EM is used to determine electron densities of protein complexes (and their structures).

    Nuclear Magnetic Resonance (NMR)

    • NMR is a method used to determine the structure of proteins. This method uses a magnetic field to analyze the properties of the spins of atomic nuclei.
    • Scalar and NOE coupling are used to distinguish protons belonging to the same amino acid or those that are close in space..
    • NMR provides information about the connectivity between atoms and helps to calculate the structure of proteins and protein-ligand complexes.
    • NMR is suitable for smaller proteins (typically under 250 amino acids).
    • The signal dispersion increases relatively little with protein size. This means large proteins have a large overlap of the signals which hampers unambiguous assignment which is needed for accurate 3D structure determination.

    Protein Crystallization for X-Ray Experiments

    • Protein crystallization is vital for X-ray crystallography of proteins.
    • Factors influencing crystal formation include pH, temperature, protein concentration, protein purity, the type of solvent and precipitant including the presence of ions or ligands.

    Quality Assessment

    • Assessing structural quality involves using statistical parameters like R-factors, Rfree factors, RMSD values, Ramachandran plot analyses, and the number of restraints per residue.
    • This is crucial to determine reliability.
    • Different resolutions of the crystal structures (differing in A (ångstrom)) provide various structural features for detailed analysis.

    Additional Methods and Considerations

    • Techniques like multiwavelength anomalous dispersion (MAD) and multiple isomorphous replacement (MIR) can help resolve phase problems in X-ray crystallography.
    • Types of structural representations include wire models, ribbon models, ball-and-stick models, and surface representations.

    Comparison of X-ray Crystallography and NMR

    • X-ray crystallography requires protein crystals whereas NMR doesn't.
    • X-ray crystallography does not have a size limitation whereas NMR does (limited to smaller proteins).
    • X-Ray procedures of proteins may be too difficult for larger proteins.
    • NMR is suitable for studying dynamic processes.

    Studying That Suits You

    Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

    Quiz Team

    Related Documents

    Experimental Methods for Structure Determination PDF

    Description

    Explore the various experimental methods used to determine protein structures, including X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance. Learn about the Protein Data Bank, which contains a wealth of information on 3D protein structures and models. This quiz will enhance your understanding of how these techniques contribute to structural biology.

    More Like This

    Structural Genomics and Protein Structure Determination
    64 questions

    Structural Genomics and Protein Structure Determination

    CourtlyDouglasFir avatar
    CourtlyDouglasFir
    Protein Tertiary Structure Quiz
    95 questions

    Protein Tertiary Structure Quiz

    SafePythagoras avatar
    SafePythagoras
    Protein Structure and Bonds
    22 questions

    Protein Structure and Bonds

    DetachableHydra avatar
    DetachableHydra
    Proteinbestimmung und -struktur
    58 questions

    Proteinbestimmung und -struktur

    ComprehensiveConceptualArt avatar
    ComprehensiveConceptualArt
    Use Quizgecko on...
    Browser
    Open
    Browser
    Browser